Deficits in cerebellum-dependent learning and cerebellar morphology in male and female BTBR autism model mice

Abstract

Recently, there has been increased interest in the role of the cerebellum in autism spectrum disorders (ASD). To better understand the pathophysiological role of the cerebellum in ASD, it is necessary to have a variety of mouse models that have face validity for cerebellar disruption in humans. Here, we add to the literature on the cerebellum transgenic and induced mouse models of autism with the characterization of the cerebellum in the BTBR T+Itpr3^tf/J (BTBR) inbred mouse strain, which has behavioral phenotypes that are suggestive of ASD in patients. When we examined both male and female BTBR mice in comparison to C57BL/6J (C57) controls, we noted that both sexes of BTBR mice showed motor coordination deficits characteristic of cerebellar dysfunction, but only the male mice showed differences in delay eyeblink conditioning, a cerebellum-dependent learning task that is also disrupted in ASD patients. Both male and female BTBR mice showed considerable expansion of and abnormal foliation in the cerebellum vermis--including significant expansion of specific lobules in the anterior cerebellum. In addition, we found a slight but significant decrease in Purkinje cell density in both male and female BTBR mice, irrespective of lobule. Furthermore, there was a marked reduction of Purkinje cell dendritic spines density in both male and female BTBR mice. These findings suggest that, for the most part, the BTBR mouse model successfully phenocopies many of the characteristics of the subpopulation of ASD patients that have a hypertrophic cerebellum. We discuss the significance of strain differences in the cerebellum as well as the importance of this first effort to identify both concordances and difference between male and female BTBR mice with regard to the cerebellum.

Keywords: autism spectrum disorder; cerebellum; idiopathic; mouse model.

Figure 1

Male BTBR show motor learning and coordination deficits, while female BTBR mice show only motor coordination deficits. (A) Male BTBR mice fall earlier than male C57 mice across two days of rotarod testing. (B) Female BTBR mice fall earlier than female C57 mice across two days of rotarod testing. (C–E) Male BTBR mice lag behind C57 mice in conditioned response performance in the delay eyeblink conditioning task across twelve training days (C) with a significant difference at the end of training (D). However, there is no difference in response timing on CS trials (E). (F–H) Female BTBR mice reach comparable levels of conditioned response performance in the delay eyeblink conditioning task across twelve training days (F) with no difference in performance at the end of training (G). However, there is no difference in response timing on CS trials (H). Black, C57B6/J mice; purple, BTBR mice. Error bars denote standard error of the mean. Asterisks denote significant results from two-sample t-tests (A,B,D,F) or planned comparisons following significant effects in a two-way ANOVA (C,E). *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

Figure 5

Male and Female BTBR mice show alterations to Purkinje cell dendritic branching and spine density. (A) Representative examples of Purkinje cells from BTBR (left) and C57 (right) mice. (B) Sholl analysis shows no difference between male BTBR and C57 mice. (C) Purkinje cell bodies are similar in area in male BTBR mice. (D) Dendritic arbor height is not different between groups of male mice. (E) Male BTBR mice have fewer dendritic spines on their distal branches. (F) Sholl analysis shows a slight increase in the complexity of dendritic arbors of Purkinje cells from female BTBR mice. (G) Purkinje cell bodies are similar in area in female BTBR mice. (H) Dendritic arbor height is not different between groups of female mice (I) Female BTBR mice have fewer dendritic spines on their distal branches. Black, C57B6/J mice; purple, BTBR mice. Error bars denote standard error of the mean. Asterisks denote significant results from two-sample t-tests (C–E) or planned comparisons following a significant two-way ANOVA (B). **, p < 0.01; ****, p < 0.0001.

Figure 2

Male BTBR mice show vermal enlargement and foliation that varies by lobule. (A) Brain weight is comparable between strains. (B) Representative image of gross anatomical differences between C57 (left) and BTBR (right) sagittal vermis sections. Arrows identify additional lobules in the BTBR section. (C) Area of the midline vermal section is significantly larger in BTBR mice. (D) Molecular cell layer (MCL) and granule cell layer (GCL) are significantly enlarged in the BTBR vermis. (E) The number of folia in the vermis is significantly different in BTBR mice. (F) Enlargement of vermis area in BTBR mice depends on lobule. (G) Enlargement of area of the molecular cell layer in BTBR mice depends on lobule. (H) Enlargement of area of the granule cell layer in BTBR mice depends on lobule. (I) Abnormal foliation in BTBR mice depends on lobule. Black, C57B6/J mice; purple, BTBR mice. Error bars denote standard error of the mean. Asterisks denote significant results from two-sample t-tests (A–E) or planned comparisons following a significant two-way ANOVA (F–I). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3

Female BTBR mice show vermal enlargement and foliation that varies by lobule. (A) Brain weight is comparable between strains. (B) Representative image of gross anatomical differences between C57 (left) and BTBR (right) sagittal vermis sections. Arrows identify additional lobules in the BTBR section. (C) Area of the midline vermal section is significantly larger in BTBR mice. (D) Molecular cell layer (MCL), granule cell layer (GCL), and white matter areas are all significantly enlarged in the BTBR vermis. (E) The number of folia in the vermis is significantly different in BTBR mice. (F) Enlargement of vermis area in BTBR mice depends on lobule. (G) Enlargement of area of the molecular cell layer in BTBR mice depends on lobule. (H) Enlargement of area of the granule cell layer in BTBR mice depends on lobule. (I) Abnormal foliation in BTBR mice depends on lobule. Black, C57B6/J mice; purple, BTBR mice. Error bars denote standard error of the mean. Asterisks denote significant results from two-sample t-tests (A–E) or planned comparisons following a significant two-way ANOVA (F–I). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 4

BTBR mice of both sexes have slight, global decreases in vermal Purkinje cell density. (A) Representative images of calbindin-stained Purkinje cells in male BTBR and C57 mice. (B) Lobule-by-lobule analysis shows a broad decrease in male BTBR mice that is not lobule-specific. (C) Lobule-by-lobule analysis shows a road decrease in female BTBR mice that is not lobule-specific. Black, C57B6/J mice; purple, BTBR mice. Error bars denote standard error of the mean. Asterisks denote main effect of strain. *, p < 0.05; **, p < 0.01.